The goal of this research program is to understand clathrin biochemistry in order to elucidate how clathrin- coated vesicle (CCV) formation and function is regulated in cells. CCVs implement the fundamental cellular membrane traffic pathways of endocytosis and lysosome biogenesis. CCVs also participate in specialized protein sorting pathways in specific tissues, influencing physiological pathways that play critical roles in human health. These include nutrient and cholesterol uptake, glucose transporter and insulin targeting, immune receptor traffic during antigen presentation and synaptic vesicle protein recapture. The characteristic lattice structure of the CCV coat that is responsible for protein sorting is formed by interactions between clathrin heavy chains (CHCs). Clathrin light chain (CLC) subunits mediate CCV binding to Hip proteins (Hip1 and Hip1R), which can bind actin and cortactin and thereby regulate actin dynamics in the vicinity of the clathrin lattice. Work from the past funding period has defined through structural and biochemical studies how CLCs also contribute to regulation of clathrin lattice formation. Now that these biochemical properties of CLCs have been established, it remains to be determined how they influence clathrin function in cells and tissues and to discover the unique functions of the two different forms of CLC (LCa and LCb). Understanding these aspects of CLC function will be the main focus for the next funding period, as this is a remaining frontier for establishing molecular mechanisms that control specificity of clathrin pathways and diversify their cellular function. Experiments to improve structural characterization of CCV proteins and analysis of how signaling regulates CHC function will be continued from the last funding period. With these goals, the three specific aims for the next funding period are as follows. Aim 1 is to determine the molecular mechanisms for intrinsic control of clathrin function through further structural analysis of clathrin and Hip proteins and through biochemical and cellular analysis of clathrin heavy chain phosphorylation. Aim 2 is to elucidate the role of clathrin light chains and their interaction with Hip proteins in cellular functions, including migration. Aim 3 is to establish the relative roles of the two vertebrate clathrin light chains LCa and LCb at the tissue level through targeted gene deletion from mice. The experimental approaches to these studies include protein crystallography and structure-based mutagenesis, RNA interference, membrane traffic and migration assays, and gene targeting strategies to produce animals that can be conditionally depleted of LCa or LCb in specific tissues, followed by analysis of immune function and neurological function. These studies continue to pursue our long-term strategy for understanding complex cellular pathways of clathrin function that are relevant to human health and disease through applying knowledge of clathrin biochemistry to focused studies of cells, tissues and organisms.